1. Field of the Invention
The present invention relates to a gene analysis technique. More specifically, the present invention relates to a nucleic acid amplification device in which target molecules to be analyzed, such as mRNA, are separately amplified on the surfaces of solid phases such as beads in a manner such that the utilization of the target molecules is improved and mixing or excessive amplification of the target molecules can be resolved.
2. Background Art
For a gene sequence analysis technique, amplification of a target molecule to be analyzed, such as mRNA or a DNA fragment in a state in which the sequence information is preserved therein, is an important technique. One example thereof is a sequence analysis system using pyrosequencing (NATURE, Vol. 437, pp. 376-380 (2005)). In such system, it is necessary for target molecules to be separately amplified such that one type of a target molecule to be analyzed is immobilized on the surface of a single bead.
Meanwhile, as an elemental technique for amplification in a separate manner, there exists a method involving separation with the use of aqueous micelles in oil (PNAS, Vol. 100, No. 15, pp. 8817-8822 (2003)). In this method, stirring of two phases (an aqueous phase and an oil phase) is performed to form aqueous micelles each retaining a set of a bead, a target molecule, a reagent necessary for amplification of nucleic acid, and the like, and then nucleic acid amplification is carried out on the bead surface with a thermal cycle. Ideally, a single micelle should contain a single bead and a single target molecule. However, it is impossible to achieve such conditions for every micelle. Thus, a micelle containing a bead but no target molecule or a micelle containing a target molecule but no bead could exist. Furthermore, a micelle containing a plurality of beads and/or target molecules or a micelle containing neither bead nor target molecule could exist (PNAS, Vol. 100, No. 15, pp. 8817-8822 (2003)). In a case in which a single micelle contains a plurality of target molecules, a mixture of target molecules is generated upon amplification, making sequence analysis impossible. In addition, in a case in which a micelle contains a target molecule but no bead, the target molecule cannot be amplified on the bead surface, resulting in loss of the target molecule. Further, in a case in which a micelle contains a plurality of beads, amplification derived from an identical target molecule is carried out on the surfaces of a plurality of beads, resulting in excessive analysis, which is problematic.
Furthermore, an amplification method wherein an assembly region (colony) is generated on a part of the flat plate surface of a glass slide or the like has been reported (Cell, Vol. 129, pp. 823-837 (2007)). According to this method, a flat plate having amplification primers immobilized thereon is used to cause complementary strand binding between the primers on the flat plate and target molecules, provide that the concentration distribution of the molecules is determined in a manner such that a certain distance is secured between the target molecules. Then, primer elongation and complementary strand binding (bridge type) are repeatedly induced with the use of primers surrounding each target molecule, such that amplified product colonies are formed. The method is problematic in that excessive amounts of target molecules are necessary due to low efficiency of complementary strand binding of primers to a plane face, and in that complementary strand binding of a plurality of neighboring target molecules might cause binding of amplified product colonies, resulting in mixing of amplified products.